Dataset Information

In Vitro and In Vivo Antiviral Activity of Nylidrin by Targeting the Hemagglutinin 2-Mediated Membrane Fusion of Influenza A Virus.

ABSTRACT: Influenza A virus, one of the major human respiratory pathogens, is responsible for annual seasonal endemics and unpredictable periodic pandemics. Despite the clinical availability of vaccines and antivirals, the antigenic diversity and drug resistance of this virus makes it a persistent threat to public health, underlying the need for the development of novel antivirals. In a cell culture-based high-throughput screen, a ?2-adrenergic receptor agonist, nylidrin, was identified as an antiviral compound against influenza A virus. The molecule was effective against multiple isolates of subtype H1N1, but had limited activity against subtype H3N2, depending on the strain. By examining the antiviral activity of its chemical analogues, we found that ifenprodil and clenbuterol also had reliable inhibitory effects against A/H1N1 strains. Field-based pharmacophore modeling with comparisons of active and inactive compounds revealed the importance of positive and negative electrostatic patterns of phenyl aminoethanol derivatives. Time-of-addition experiments and visualization of the intracellular localization of nucleoprotein NP demonstrated that an early step of the virus life cycle was suppressed by nylidrin. Ultimately, we discovered that nylidrin targets hemagglutinin 2 (HA2)-mediated membrane fusion by blocking conformational change of HA at acidic pH. In a mouse model, preincubation of a mouse-adapted influenza A virus (H1N1) with nylidrin completely blocked intranasal viral infection. The present study suggests that nylidrin could provide a core chemical skeleton for the development of a direct-acting inhibitor of influenza A virus entry.

SUBMITTER: Jang Y

PROVIDER: S-EPMC7290441 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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In Vitro and In Vivo Antiviral Activity of Nylidrin by Targeting the Hemagglutinin 2-Mediated Membrane Fusion of Influenza A Virus.

Jang Yejin Y Shin Jin Soo JS Lee Joo-Youn JY Shin Heegwon H Kim Sang Jick SJ Kim Meehyein M

Viruses 20200525 5

Influenza A virus, one of the major human respiratory pathogens, is responsible for annual seasonal endemics and unpredictable periodic pandemics. Despite the clinical availability of vaccines and antivirals, the antigenic diversity and drug resistance of this virus makes it a persistent threat to public health, underlying the need for the development of novel antivirals. In a cell culture-based high-throughput screen, a β2-adrenergic receptor agonist, nylidrin, was identified as an antiviral co ...[more]

PMID: 32466302

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Project description:The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion.IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion.

Project description:The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins.IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans.

Dataset Information

In Vitro and In Vivo Antiviral Activity of Nylidrin by Targeting the Hemagglutinin 2-Mediated Membrane Fusion of Influenza A Virus.

Publications

In Vitro and In Vivo Antiviral Activity of Nylidrin by Targeting the Hemagglutinin 2-Mediated Membrane Fusion of Influenza A Virus.

Similar Datasets

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